Healing an epidermal wound requires the coordination of tissues and extracellular matrix (ECM), and this coordination is mediated by signaling pathways. Matrix metalloproteinases (MMPs) are proteases that cleave ECM and regulate signaling pathways in vitro, in culture, and during development, but their roles in wound healing are unclear. MMPs are medically important: they are highly upregulated during tumor progression, inflammation, and wound healing. We use a Drosophila model of epidermal wound healing to elucidate MMP function because of its reduced complexity - for example, there are 24 MMPs in mouse, whereas there are only two MMPs in Drosophila, Mmp1 and Mmp2. This organism also offers powerful genetic tools that allow spatio- temporally controlled gain- and loss-of-function phenotypic analyses for nearly every gene. We hypothesize that the Drosophila MMPs form a complex that acts as a global regulator of basement membrane remodeling. This hypothesis is based on genetic analyses of Mmp1, Mmp2, and the MMP binding-protein Timp in wounding and developmental contexts. All are required for healing puncture wounds, for specific aspects of pupal morphogenesis, and for expanding basement membrane during normal growth. Although these genes have other independent phenotypes, the shared phenotypes all point to a fundamental role for the three genes in basement membrane remodeling. As an MMP-Timp trimolecular complex has been described in mammalian cell culture, we hypothesize that Mmp1, Mmp2, and Timp form a similar complex that directs expansion and repair of basement membranes. Supporting this hypothesis, Mmp1's normal localization to the basement membrane and wound margins is lost in Mmp2 and Timp mutants. This hypothesis is novel and exciting: although MMPs are known to cleave ECM, they are considered to degrade ECM, rather than promote its expansion and repair. The MMPs also appear to regulate ERK signaling at the wound margin, with Mmp1 promoting signaling and Mmp2 confining signaling to the margin. These opposing phenotypes suggest another function of the same MMP complex. Aim 1 determines the molecular interactions among Mmp1, Mmp2 and Timp, testing the MMP-complex hypothesis. We will identify binding partners, determine if they co-localize in tissues, and if different Mmp1 splice forms have different partners. Aim 2 determines how MMPs individually and together modify basement membrane, identifying substrates by biochemical methods and an innovative proteomics approach (iTRAQ- TAILS); identifying the tissues controlling remodeling with conditional genetics; and examining the fine structure of mutant basement membrane. Aim 3 determines how the two MMPs oppositely regulate ERK signaling at wounds in vivo, identifying the receptor and ligand, determining the function of ERK signaling in wound healing; and identifying the specific MMP substrate(s) responsible for altering the pathway. To accomplish these aims we have garnered support from a broad team of experts to inform and guide our experiments. PUBLIC HEALTH RELEVANCE: Wound healing is important to human health, and millions of people are affected by chronic ulcerating wounds that do not heal or by disfiguring scars resulting from wound healing. We will examine how families of proteases that are thought to be destructive actually contribute to wound healing. Our findings will also be relevant for cancer patients, as the same family of proteases is present at high levels in tumors.